Eggenreich K, Golouch S, Töscher B, Beck H, Kuehnelt D, Wintersteiger R
Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens-University, Schubertstrasse 1 A-8010, Graz, Austria.
J Biochem Biophys Methods. 2004 Oct 29;61(1-2):23-34. doi: 10.1016/j.jbbm.2004.04.021.
A multi-step gradient HPLC system combined with DAD and MS detection has been developed for the determination of the oxidation hair dyes 4-amino-m-cresol (4-AC) and 5-amino-o-cresol (5-AC) and their metabolites in the alternative testing system human keratinocytes (HaCaT) cell culture. The culture medium induced by 3-methylcholanthrene (3-MC) was fortified with 4-AC or 5-AC and incubated for 24 h at 37 degrees C in order to produce metabolites. After several pre-cleaning steps, further cleaning was done by solid-phase extraction using C18 phenyl cartridges. Optimizing chromatographic conditions, a hybrid-based RP8 column was most suitable for the separation of the metabolites formed in HaCaT. Only one conjugation product, the N-acetylated derivative, could be identified for both 4-AC and 5-AC by LC/DAD/MS. The ionisation technique used for MS analysis was Atmospheric Pressure Ionization (API).
已开发出一种结合二极管阵列检测器（DAD）和质谱（MS）检测的多步梯度高效液相色谱系统，用于在替代测试系统人角质形成细胞（HaCaT）细胞培养物中测定氧化染发剂4-氨基间甲酚（4-AC）和5-氨基邻甲酚（5-AC）及其代谢物。用4-AC或5-AC强化由3-甲基胆蒽（3-MC）诱导的培养基，并在37℃下孵育24小时以产生代谢物。经过几个预清洗步骤后，使用C18苯基柱进行固相萃取进一步清洗。优化色谱条件后，基于混合的RP8柱最适合分离HaCaT中形成的代谢物。通过LC/DAD/MS仅能鉴定出4-AC和5-AC的一种共轭产物，即N-乙酰化衍生物。用于MS分析的电离技术是大气压电离（API）。
